Saudi Journal of Biological Sciences 28 (2021) 5204–5213

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Saudi Journal of Biological Sciences

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Original article

A new cerebral ischemic injury model in rats, preventive effect of gallic

acid and in silico approaches
P. Praveen Kumar a,⇑, Madhuri D. b, L. Siva Sankar Reddy a, Y. Dastagiri Reddy a, G. Somasekhar c,
N.V.L. Sirisha d, K. Nagaraju e, M.S. Shouib b, A.S. Rizwaan b
a
Santhiram College of Pharmacy, Nandyal, Kurnool, Andhra Pradesh, India
b
Creative Educational Societys College of Pharmacy, Kurnool, Andhra Pradesh, India
c
SKU College of Pharmaceutical Sciences, Anantapur, Andhra Pradesh, India
d
Nitte College of Pharmaceutical Sciences, Banglaore, Karnataka, India
e
C.R Reddy College of Pharmacy, Eluru, West Godavari, Andhra Pradesh, India

a r t i c l e i n f o a b s t r a c t

Article history: Current study was designed multiple occlusions and reperfusion of bilateral carotid arteries induced cere-
Received 2 April 2021 bral injury model and evaluated the protective effect of gallic acid on it. In silico study was involved to
Revised 17 May 2021 study gallic acid binding affinity on cerebrotonic proteins compared with standard drugs using
Accepted 18 May 2021
Autodoc vina tool. Cerebral ischemia was induced by occlusion of bilateral common carotid arteries for
Available online 24 May 2021
10 mins followed by 10 reperfusions (1 cycle), cycle was continued to 3 cycles (MO/RCA), then patholog-
ical changes were observed by estimation of brain antioxidants as superoxide dismutase, glutathione,
Keywords:
catalase, oxidants like malonaldehyde, cerebral infarction area, histopathology, and study gallic acid
GA (Gallic acid)
Multiple occlusion-reperfusion of bilateral
treatment against cerebral injury. Gallic acid exhibited a strong binding affinity on targeted cerebrotoxic
common carotid arteries (MO/RCA) proteins. MO/RCA rat brain antioxidant levels were significantly decreased and increased MDA levels
In silico (p < 0.0001), Infarction size compared to sham rats. Gallic acid treatment rat brain MDA levels signifi-
Antioxidants cantly decreased (p < 0.4476) and increased SOD (p < 0.0001), CAT (p < 0.0001), GSH (p < 0.0001), cerebral
Inflammatory mediators infarction area when compared to MO/RCA group. Developed model showed significant cerebral ischemic
injury in rats, injury was ameliorated by Gallic acid treatment and in silico approaches also inhibit the
cerebrotoxic protein function by targeting on active sites.
Ó 2021 The Author(s). Published by Elsevier B.V. on behalf of King Saud University. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction
Throughout the world, Currently Ischaemic stroke (IS) is a cere-
brovascular disease with high range of severity in morbidity and
mortality (Feigin et al., 2016). The occurrence of this disease is of
2 varieties i.e. focal & global. When the amount of blood flow is less
or decrease in flow of blood to specific regions of brain-embolic
middle cerebral artery occlusion (MCAO) is known as Focal ische-
mia. global ischemia happens when cerebral blood flow (CBF) is
reduced throughout the parts of brain which in turn leads to car-
⇑ Corresponding author at: Head of the Department of Pharmacology, Santhiram
College of Pharmacy, Nandyal, Kurnool, Andhra Pradesh, India.
E-mail address: praveenpharmaco@gmail.com (P. Praveen Kumar).
Peer review under responsibility of King Saud University.
Production and hosting by Elsevier
diac arrest (Smith, 2004; Traystman, 2003). The tissue damage
occurs when there is a back flow of blood to the tissues, Cerebral
ischemia–reperfusion injury (CIRI) which is very common in
ischemic stroke. CIRI persuades oxidative stress which in turn trig-
gers neuronal loss and cognitive impairment, circulation regenera-
tion results in inflammation, and harmful oxidation effects (Ritzel
et al., 2015), hence inflammatory agents play a key role in damage
of ischemic brain tissue (Liu et al., 2018) along with antioxidant
enzymes to modulate and provoke neuronal cell defence against
toxic reactive oxygen species (ROS) (Ding et al., 2015; Farrell-
Dillon et al., 2017) with this justification antioxidants have been
advised in vitro and in vivo for a desirable pursuit for CIRI therapy,
as the crucial role was played by oxidative stress. Globally 11% of
total deaths are happened due to stroke when compared to all
life-threatening diseases, especially in India the main cause for
death is stroke according to the epidemiological statistics
(Banerjee and Das, 2016; Kamalakannan et al., 2017) and cere-
brovascular diseases are considered the second most common
cause of expected deaths in 2020 (Huang and McNamara, 2004).

https://doi.org/10.1016/j.sjbs.2021.05.044
1319-562X/Ó 2021 The Author(s). Published by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
P. Praveen Kumar, M. D., L. Siva Sankar Reddy et al.
In the current scenario we must pay attention to the management
of ischemic brain damage with relevant research findings
(O’Rourke, 2004). Among the phytoactives, Gallic acid (GA), 3,4,5-
trihydroxybenzoic acid, was more focussed by researchers, with
its unique and potential constituents present in various parts of
plants and different fruits like grapes (Cedó et al., 2014; Scoccia
et al., 2016; Yang et al., 2020), gallnuts (Liu et al., 2019; Wang
and Li, 2017), pomegranates (Singh et al., 2014; Zhang et al.,
2018), and tea leaves (Jiang et al., 2019). To be specific, GA and
derivatives are used as flavouring agents and preservatives in food
industry (Alfei et al., 2019; Fereidoonfar et al., 2019) also it has
many pharmaceutical applications as antioxidants (Phonsatta
et al., 2017; Wang et al., 2019), antimicrobial (Wang et al., 2019)
(Rosman et al., 2018), anticancer (T. Zhang et al., 2019), anti-
inflammatory, gastroprotective (Pandurangan et al., 2015), cardio-
protective (El-Hussainy et al., 2016), neuroprotective (Maya et al.,
2018b), in addition to the usage in prevention of metabolic dis-
eases (Bak et al., 2013; Huang et al., 2016). The current study is
more focussed antioxidant/oxidant status and infarction size in
MO/RCA and evaluate the protective effect of Gallic acid on injury
in rats.
2. Materials and methods
2.1. Drugs and chemicals
Gallic acid, 2,3,5-Triphenyl tetrazolium chloride (TTC),
5,50 -dithiobis-(2- nitrobenzoic acid) (DTNB), NADH, Phenazine
methosulphate, were purchased from Sisco research laboratories
Pvt. ltd. Sodium lauryl sulphate, Glacial acetic acid, dipotassium
hydrogen phosphate, Tris buffer were purchased from Fisher
scientific. Trichloro acetic acid, Sodium pyro phosphate, H2O2 were
purchased from S D Fine-Chem Limited. N-butanol, Pottassium
dihydrogen phosphate were purchased from Merck Life Science
Pvt Ltd. All other chemicals were of the highest purity
commercially available.
2.2. Docking studies
2.2.1. Experimental procedure of binding energy between Gallic acid
and neurotoxic proteins by using PyRx tool
Tumor necrosis factor a (Dong et al., 2017), Caspases 3 and Bax
(Chaitanya and Babu, 2008), Nitric oxide synthase (Chen et al.,
2017), Interleukin 6 (Aref et al., 2020), Glutamate receptors (Sun
et al., 2019) and Acid sensing ion channel (Dibas et al., 2018) acti-
vation involved in disease progression. The virtual screening was
performed by using PyRx virtual screening tool between Gallic acid
and above cerebrotoxic proteins which are involved in cerebral
stroke they are like Bax (PDB ID: 5 W60), Tumor necrosis factor
a (PDB ID : 5UUI), Caspases 3 (PDB ID : 2DKO), Interleukin (PDB
ID : 1ALU), Nitric oxide synthase (PDB ID : 6CIC) compared to stan-
dard drugs such as Minocycline (Pub Chem Id : 54675783), Quer-
cetin (Pub Chem Id:5280343) with Acid sensing ion channel (PDB
ID : 3S3X); Memantine (Pub Chem Id: 4054) with Glutamate recep-
tor (Pub Chem Id:5ZG2). Minocycline (Naderi et al., 2020), Querce-
tin (Wang et al., 2020), Memantine (Tanaka et al., 2018) reported
significant neuroprotective effect on targeting reported cerebro-
toxic proteins. Hence current study was using these compounds
as standard. Discovery studio2017 R2 tool for visualizing protein
and drug interaction and Autodoc vina tool used for binding affin-
ity toward neurotoxicity proteins. Here we downloaded proteins
3D structure from the PDB database using PDB codes. Then deleted
ligand groups, water molecules, and heteroatom and applied force
field in protein molecule using BIOVIA Discovery studio. The pro-
tein was later saved for virtual screening. Minimized protein and
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Saudi Journal of Biological Sciences 28 (2021) 5204–5213
ligand converted to PDBQT, selected maximized GRID parameter
then performed docking study (Trott and Olson, 2009). Highest
negative binding energy indicates good stability at binding site.
The model with highest negative binding energy compound and
protein complex was selected and visualized protein and ligand
type of interaction and distance using BIOVIA Discovery stu-
dio2017 R2 tool.
2.2.2. Prediction of protein active site
Active sites of Caspases 3, identified as HIS A: 121, ARG B: 207
CYS A: 163, SER A: 58, TYR B: 204. Active sites of Interleukin 6,
identified as GLN A: 156, ARG A: 40, LYS A: 171 GLN: 175. Active
sites of Nitric oxide synthase, identified as PHE B: 709, ALA B:
417, ARG B: 419, ALA A: 417, CYS A: 420. Active sites of Glutamate
receptor, identified as THR A: 501, PRO A: 499, GLU A: 726, TYR A:
471, PRO A: 515, ARG B: 506. All active sites were predicted by
using Bio via Drug discovery studio visualizer 2017. After loading
the protein structure to DSV, it reads the protein and highlights
the probable active site, i.e., residue information in yellow colour
(Design LI, 2014).
2.3. Animals
Adult wistar rats (220–250 g) either sex was procured from Sai-
nath agency Pvt. Ltd., Hyderabad, Telangana, India and maintained
under a 12/12-h light/dark cycle, in an ambient room temperature
(24 ± 1 °C). Animals were provided with an adequate supply of food
and water. Study protocol approved by IAEC/CESCOP/2019-OCT-
02.
2.4. Experimental protocol for cerebral ischemia
Rats were divided into five groups; each group contain six rats.
Group I Normal rats
Group II Sham rats
Group III Disease rats (MO/RCA)
Group IV Pre-treatment of GA (25 mg/kg B.Wt. i.p) for 10 days
followed by MO/RCA surgery.
Group V Pre-treatment of GA (50 mg/kg B.Wt. i.p) for 10 days
followed by MO/RCA surgery.
2.5. Induction of cerebral ischemic rats by MO/RCA model
Ketamine (60 mg/kg,), xylazine (10 mg/kg) was used to anes-
thetize rats. Both common carotid arteries left, and right were
carefully separated and maintained from all muscles, ligaments,
their adventitial sheath, and vagus nerve. Both common carotid
arteries occluded 10 mins, 10 reperfusion (1 cycle), cycle was con-
tinued to 3 cycles (MO/RCA). By using waxed silk suture, the skin
was closed. Dilated pupils, absence of cornea reflex on light expo-
sure and rectal temperature maintenance at 37 ± 0.5 °C observed
after completion of 3rd cycles. Hypothermia development been
prevented using a heating lamp on the surgical table.
After completion of 3 cycles, rats were euthanized, isolated the
brain, washed in cooled 0.9% saline, then estimated cerebral infarc-
tion size using TTC staining method (Bederson et al., 1986). Brain
was homogenized as 10% (w/v) in cold phosphate buffer (0.05 M,
pH 7.4). The homogenates were centrifuged at 1000  g for
10 min at 4◦C. Supernatant were subjected for estimation of esti-
mation of glutathione (GSH) (Ellman, 1959) superoxidase dismu-
tase (SOD) (Kono, 1978) and malondialdehyde (MDA) (M Stefan
and Gudrun, 1974) and Catalase (A Claibome, 1985).
P. Praveen Kumar, M. D., L. Siva Sankar Reddy et al. Saudi Journal of Biological Sciences 28 (2021) 5204–5213

2.6. Histological studies Table 1

Type of interaction and interaction distance between Gallic acid on Neurotoxic
proteins.
Sagittal brain sections were fixed in freshly prepared 10%
neutral buffered formalin and embedded in paraffin. In addition, S. Protein PyRx Type of interaction with residue (Interaction
4-lm-thick paraffin sections were prepared and stained with No Name Kcal/mol Distance)

hematoxylin and eosin (H&E) for histopathological examination. 1 Bax 5.6 Hydrogen bonding ASPA: 53 (2.25 Å), SER
Sections were examined using a light microscope (Tabassum A: 55 (2.49 Å), THR A :56 (2.68 Å), TRP A:
158 (2.73 Å), PRO A: 13 (2.93 Å)
et al., 2013). 2 Tumor 5.6 Hydrogen bonding GLU A: 149 (1.85 Å),
necrosis VAL A: 150 (1.52 Å), GLY A: 148 (1.62 Å),
factor a ALA A: 18 (2.63 Å)
2.7. Statistical method
Pi alkyl bonding ARG A: 32 (3.62 Å)
Pi sigma bonding VAL A: 17 (4.26 Å)
The mean ± SEM were calculated for all data significant differ- 3 Caspases 3 5.5 Hydrogen bonding THR A: 77 (1.91 Å), ASP
ent between means where evaluated by t-test and one-way Analy- B: 228 (1.86 Å), LYS B: 224 (2.64 Å)
sis Variance (ANOVA followed by Dunnet comparison test p < 0.05 Pi Alkyl bonding ALA B: 227 (3.36 Å), LEU
A: 81 (4.50 Å)
considered as statistically significant. 4 Nitric oxide 6.7 Hydrogen bonding PHE B: 709 (1.88 Å),
synthase ARG B: 704 (1.90 Å), SER B :418 (2.23 Å)
Pi sigma bonding ALA B: 417 (2.67 Å)
3. Results Pi alkyl bonding ARG B: 419 (4.38 Å)
Pi Pi T Shaped HIS B: 346 (4.05 Å)
3.1. Docking analysis 5 Interleukin 6 5.9 Hydrogen bonding SER A: 107 (1.89 Å),
GLU A: 42 (2.90 Å), ARG A: 104 (2.45 Å), ASP
A: 160 (2.23 Å)
3.1.1. Binding affinity on Bax Pi alkyl bonding PHE A: 105 (4.89 Å)
Gallic acid was seen to form hydrogen bonds with ASP A: 53, 6 Glutamate 5.8 Hydrogen bonding ARG A: 506 (1.80 Å),
TRP A: 158, PRO A: 13 having interaction distance 1.62 Å, 1.92 Å, receptors THR A: 501 (2.91 Å), PRO A :499 (2.29 Å)
2.62 Å respectively and exhibited binding energy 5.6 K Cal/mol Pi Anion bonding GLU A :726 (3.16 Å)
Pi Pi Stacked TYR A : 471 (4.15 Å)
(Fig. 1) (Table 1).
7 Acid sensing 6.7 Hydrogen bonding GLN C: 279 (2.03 Å),
Minocycline was exhibited hydrogen bonds with ASP A: 98, ASP ion channel ARG C: 370 (2.15 Å), ARG B: 370 (2.23 Å),
A: 102 having interacting distance of 1.90 Å, 2.03 Å respectively. GLY N A: 277 (2.62 Å), GLU A: 417 (2.764 Å),
Amide pi Stacked with GLY A: 179 having interacting distance of GLU B: 47 (2.95 Å)
3.89 Å, Pi alkyl with VAL A: 180 having interacting distance of
4.05 Å and exhibited binding energy 6.2Kcal/mol (Fig. 1; Table 2).
Minocycline was seen to form hydrogen bonds with HIS A: 121,
ARG B: 207, CYS A: 163 having interacting distance of 1.55Å, 2.22 Å
3.1.2. Binding affinity on Tumor necrosis factor a protein
2.52Å respectively and exhibited binding energy 7.4Kcal/mol
Gallic acid was seen to form hydrogen bonds with GLN A: 149,
(Fig. 3; Table 2).
VAL A: 150, ALA A: 18, GLY A: 150 having interacting distance 1.74
Å, 1.85 Å, 2.38 Å, 2.18 Å respectively. Pi sigma with VAL A: 17 hav-
ing interacting distance 3.05 Å, Pi alkyl with ARG A: 32 having
3.1.4. Binding affinity on Nitric oxide synthase
interacting distance 4.35 Å and exhibited binding energy –5.6
Gallic acid was exhibited hydrogen bonds with PHE B: 709, ARG
Kcal/mol (Fig. 2; Table 1).
B: 706, SER B: 418 having interacting distance of 1.53 Å, 2.02 Å,
Minocycline was showed hydrogen bonds with ASP A: 140, LYS
2.57 Å respectively. Pi sigma ALA B: 417 having interacting dis-
A : 65, PHE A : 144, PRO A : 20 having interacting distance of 1.93 Å,
tance of 3.62 Å. Pi Pi T Shaped with HIS B: 346 having interacting
1.95 Å, 2.19 Å, 2.35 Å and exhibited binding energy 7.1Kcal/mol
distance of 3.65 Å. Pi alkyl with ARG A: 419 having interacting dis-
(Fig. 2; Table 2).
tance of 4.62 Å and exhibited binding energy 6.7Kcal/mol (Fig. 4;
Table 1).
3.1.3. Binding affinity on Caspases 3 Minocyclin was seen to form hydrogen bonds with TRP B: 592,
Gallic acid was observed to form hydrogen bonds with ASP B: GLU B: 597 having interacting distance of 1.85 Å, 2.85 Å respec-
228, THR A: 77, LYS B:224 having interaction distance 1.32 Å, tively. Pi alkyl with VAL B: 572, MET B: 575 having interacting dis-
1.23 Å, 1.62 Å respectively. Pi alkyl with LEU A: 81, ALA B: 227 hav- tance of 3.98 Å, 4.25 Å respectively. Alkyl with PHE B: 589 having
ing interacting distance of 3.22 Å, 4.23 Å respectively and exhibited interacting distance of 4.32 Å and exhibited binding energy 8.8K-
binding energy –5.5 Kcal/mol (Fig. 3; Table 1). cal/mol (Fig. 4; Table 2).

Fig. 1. Binding affinity on Bax Protein.

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Table 2 tion distance 2.52 Å, Pi anion with GLU A: 726 having interaction
Type of interaction and interaction distance between Minocycline and neurotoxicity distance 2.95 Å and exhibited binding energy 5.8Kcal/mol (Fig. 7;
proteins.
Table 1). Memantine observed to form hydrogen bonds with GLN
S. Protein PyRx Type of interaction with residue B: 663, SER B: 661 having interacting distance of 2.25 Å, 2.56 Å
No Name Kcal/mol (Interaction Distance) respectively. Pi alkyl with TYR B: 694, LYS B: 690 with having inter-
1 Bax 6.2 Hydrogen Bonding ASP A: 98(1.90 Å), ASP acting distance of 3.62 Å, 4.22 Å respectively and exhibited binding
A: 102(2.03 Å) energy 5.6Kcal/mol (Fig. 7; Table 4).
Amide pi Stacked GLY A: 179(3.89 Å)
Pi alkyl VAL A: 180(4.05 Å)
2 Tumour 7.1 Hydrogen Bonding ASP A: 140 (1.93 Å), LYS
necrosis A: 65 (1.95 Å), PHE A: 144 (2.19 Å), PRO A: 3.2. Cerebral infarction area
factor a 20 (2.35 Å)
3 Caspases 3 7.4 Hydrogen Bonding HIS A: 121 (1.55 Å),
Gallic acid (25 mg/kg, 50 mg/kg, B.wt) treated rats the percent
ARG B: 207 (2.22 Å), CYS A: 163 (2.52 Å)
4 Nitric oxide 6.6 Hydrogen Bonding TRP B: 592 (1.85 Å), cerebral infarct volumes were found to be reduced. In this study,
synthase GLU B: 597 (2.85 Å) there was a significant increase in percent cerebral infarction in
Pi alkyl bonding VAL B: 572 3.98 Å), MET B: MO/RCA group compared to normal control group. Treatment with
575 4.25 Å)
Gallic acid significantly reduced in percent cerebral infarction com-
Alkyl bonding PHE B: 589 (4.32Å), CYS B:
420 (4.52 Å) pared to MO/RCA group, indicating the cerebroprotective action of
5 Interleukin 6 8.8 Hydrogen Bonding ARG A: 104 (1.95 Å), Gallic acid (Figs. 8, 9; Table 5).
GLN A: 156 (2.55 Å), GLU A: 106 (3.98Å)
Pi sigma bonding PHE A: 105 (4.58 Å)

3.3. Effect of Gallic acid on biochemical parameters in MO/RCA rats

Results were revealed that potential neuroprotective activity of

3.1.5. Binding affinity on Interleukin 6
Gallic acid was showed to form hydrogen bonds with SER
A:107, GLUA: 42, ARG A: 104, ASP A: 160 having interaction dis-
tance 1.80 Å, 1.12 Å, 2.23 Å, 2.12 Å respectively. Pi-Pi shaped with
PHE A :105 having interacting distance of 3.12 Å and exhibited
binding energy 5.9Kcal/mol (Fig. 5; Table 1). Minocycline was
observed to form hydrogen bonds with ARG A: 104, GLN A: 156,
GLU A: 106 having interacting distance of 1.95Å, 2.55 Å, 3.98 Å
respectively. Pi sigma with PHE A: 105 having interacting distance
of 4.58 Å and exhibited binding energy 6.6Kcal/mol (Fig. 6;
Table 2).
GA. MDA levels exhibited significant increase and all other enzy-
matic and non-enzymatic parameters (SOD, CAT, and GSH) showed
a significant decrease in the MO/RCA group. The animals from GA
treated groups had shown a significant protection by reducing
the elevated levels of MDA (P < 0.4476) and marked increase in
SOD (P < 0.001), CAT (P < 0.01), and GSH (P < 0.001) as compared
to MO/RCA treated group. The levels of all enzymatic and non-
enzymatic parameters were normal in normal and sham group
(Fig. 10; Table 5).
3.4. Histopathology

3.1.6. Binding affinity on acid sensing ion channel
Gallic acid was shown to form hydrogen bonds with GLN C: 279,
ARG C: 370, GLU B: 417, GLN A: 277, GLU A :417 having interaction
distance 1.50 Å, 1.56 Å, 1.62 Å, 2.52 Å, 2.72 Å respectively and
exhibited binding energy 8.0 Kcal/mol (Fig. 6; Table 1). Quercetin
was observed to form hydrogen bonds with GLU C: 98, ARG C: 191,
GLU C: 154, ARG F: 28, SER C: 241, GLU C: 243 having interacting
distance of 1.62 Å, 1.85 Å, 2.32 Å, 2.62 Å, 2.74 Å, 2.82 Å respectively
and exhibited binding energy 9.3Kcal/mol (Fig. 6; Table 3).
3.1.7. Binding affinity on Glutamate
Gallic acid was seen to form hydrogen bonds with PRO A: 499,
THR A: 501, ARG A: 506 having interaction distance 1.95 Å, 1.99 Å,
2.62 Å respectively. Pi Pi Stacked with TYR A: 471 having interac-
In this study we unveil that during cerebral ischemia cells can
lose their cell permeability barrier properties, leading to edema
formation and degradation, and the hippocampus region of the
brain showed decreased thickness of the pyramidal layer, with
increased apoptotic neurons with dystrophic changes in the form
of shrunken and irregular of MO/RCA group when compared to
normal, sham and treated groups. However, MO/RCA surgery
induced severe ischemia and neuronal damage, manifested as
decreased in the intact of neurons when compared with normal
rats and sham rats. Moreover, Gallic acid 25 mg and 50 mg/kg
treatment showed restore neuronal cells contact when compared
with MO/RCA rats. In this neuronal intact more in Gallic acid
50 mg/kg when compared with Gallic acid 25 mg/kg treatment
groups (Fig. 11).

Fig. 2. Binding affinity on Tumour necrosis factor a.

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Fig. 3. Binding affinity on Caspases 3.

Fig. 4. Binding affinity on Nitric oxide synthase.

Fig. 5. Binding affinity on Interleukin 6.

Fig. 6. Binding affinity on Acid sensing Channels.

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P. Praveen Kumar, M. D., L. Siva Sankar Reddy et al.
Table 3
Type of interaction and interaction distance between Quercetin on Acid sensing ion
channel.
S. Protein PyRx Type of interaction with residue (Interaction
No Name Kcal/mol Distance)
1 Acid sensing 9.3 Hydrogen Bonding GLU C: 98 (1.62 Å), ARG
ion channel C: 191 (1.85 Å), GLU C: 154 (2.32 Å), ARG F:
28 (2.62 Å), SER C: 241 (2.74 Å), GLU C: 243
(2.82 Å)
3.5. Histopathological studies
Fig. 11.
4. Discussion
Cerebral ischemia and reperfusion both lead to neuronal dam-
age (Jiang et al., 2010). In this study, we evaluated, for the first
time, the brain antioxidant/stress parameters and infarction size
in multiple occlusion and reperfusion of bilateral common carotid
arteries, evaluated protective effect of known neuroprotective
effective agent GA against cerebral injury. Our study demonstrated
multiple occlusion reperfusion of bilateral common carotid arteries
rats showed significantly decreased antioxidant enzymes,
increased infarct size and cerebral histological damage. Further-
more, we found that GA treatment markedly reduced MDA, as well
as oppose the decreases in SOD, catalase and GSH induced by mul-
tiple occlusion/reperfusions of bilateral common carotid arteries
induced cerebral ischemia injury rats. In silico studies was included
to assessed binding affinity on cerebrotoxic proteins in cerebral
ischemia. Cerebral damage was evaluated by the infarct size and
the histopathological changes of brain were assessed by hematoxy-
lineosin (H–E) staining in the brain slices. Our results revealed that
’’MO/RCA rats showed significant increase infarct size and histolog-
ical damage in ischemia/reperfusion mice compared to the sham
rats and protective effects of GA in a dose dependent manner.
The protective effects of GA at 50 mg/kg were more apparent than
those at 25 mg/kg, probably due to much higher brain concentra-
tions of GA at a high dose.
It is well understood that oxidative metabolism is necessary for
brain survival, but it is also correlated with the production of reac-
tive oxygen species (Madamanchi et al., 2005; Schreibelt et al.,
2007; Zhao et al., 2018). GSH, an endogenous antioxidant, is a
key element of cellular antioxidant defences since it functions both
to directly detoxify reactive oxygen species and as a substrate for
various peroxidases. Glutathione system dysfunction has been
linked to a variety of neurodegenerative diseases (Chandra
Jagetia et al., 2003; X. Zhang et al., 2019) and it can contribute to
Fig. 7. Binding affinity on Glutamate receptors Gallic acid b) Minocycline c) Quercetin d) Memantine.
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Saudi Journal of Biological Sciences 28 (2021) 5204–5213
oxidative damage following transient ischemia. Catalase is an
enzyme that scavenges harmful oxygen by products such as hydro-
gen peroxide, and a rise in catalase levels suggests antioxidant
activity. Catalase is an enzyme that converts toxic hydrogen perox-
ide to water and oxygen. Catalase activity is mainly identified in
peroxisomes, which are subcellular organelles (Weydert CJ,
2010). Inflammatory cells contain a significant amount of hydro-
gen peroxide in order to destroy pathogens. Its high concentration
is cytotoxic, and its deposition causes cellular targets such as DNA,
proteins, and lipids to oxidize, resulting in mutagenesis and cell
death. Catalase eliminates cell H2O2, thereby protecting it from
oxidative damage. SOD, another defensive system to work against
reactive oxygen species-induced tissue damage, catalyzes the dis-
mutation of superoxide anion to hydrogen peroxide and prevents
the formation of the hydroxyl radical (Cheng et al., 2019; Li
et al., 2008). MDA is a widely used oxidative stress biomarker in
several diseases, including stroke. It is a by-product of lipid perox-
idation induced by the deterioration of cellular membrane phos-
pholipids. Since MDA enters the blood after being released into
the extracellular space, it has been used as an effective diagnostic
tool of lipid oxidation (Menon et al., 2020).
Sudden bursts of reactive oxygen species cannot be managed by
endogenous antioxidant systems during ischemia/reperfusion, and
the accumulation of reactive oxygen species causes oxidative dam-
age to cellular membranes, proteins, and DNA (Thiyagarajan and
Sharma, 2004; Zheng et al., 2007). As a result, antioxidants have
been recognized as a potential therapy for ischemic stroke (J
Murdoch, 1990; Powers and Jackson, 2008). In this study we found
that the MO/RCA rats brain antioxidant enzymes GSH, catalase and
SOD were significantly decreased, MDA levels was increased com-
pared to sham rats, GA treatment markedly increased GSH, cata-
lase and SOD levels and decreased MDA levels in MO/RCA rats.
Astrogliosis is most common in neurological disorders that
cause apoptosis, neuroinflammation, and neuronal death. TNF, IL-
6, and apoptotic proteins such as caspase 8, Bax are examples of
neuroinflammatory factors formed by Glia cell activity (Jayaraj
et al., 2019; Sun et al., 2014). Neuronal death in a stroke is a com-
plicated event involving, among other things, metabolic dysfunc-
tion, excitotoxicity, loss of calcium homeostasis, and oxidative
stress (Alexi, 2000). During ischemic stroke, increased glutamate
release leads to increased Ca2+ level. The massive Ca2+ entry acti-
vates enzymes such as proteases, oxidases, phospholipases and
endonucleases that can hydrolyze the DNA molecule and destroy
the cytoskeleton (Nicotera and Lipton, 1999; Park et al., 2020;
Welch KM, 1997). Few studies have indicated that acid sensing
ion channel 1a knockout or pharmacological inhibition of acid
sensing ion channel 1a decreased infarct volume significantly
(Xiong et al., 2006). The histopathology studies revealed that in
MBCAO/R rats hippocampus region of the brain showed decreased
P. Praveen Kumar, M. D., L. Siva Sankar Reddy et al. Saudi Journal of Biological Sciences 28 (2021) 5204–5213

Table 4 infraction area was increased in MO/RCA rats as compared to con-

Type of interaction and interaction distance between Memantine on Glutamate trol groups, it was attenuated by gallic acid treatment.
receptor.
GA exhibits cerebroprotection by antioxidant and inflammation
S Protein Name PyRx Type of interaction with residue pathways in animal models of neurodegenerative diseases due to
No Kcal/mol (Interaction Distance) its susceptibility to NMDA receptors and excitotoxicity caused by
1 Glutamate 5.6 Hydrogen Bonding GLN B: 663 (2.25 Å), glutamate after cerebral ischemia accompanied by Ca+2 influx
receptors SER B: 661 (2.56 Å) and thus intracellular Ca+2 accumulation induced neuronal apopto-
Pi alkyl TYR B: 694 (3.62 Å), LYS B: 690
(4.22 Å)
sis (Mansouri et al., 2013). Antioxidative effect of GA may oppose
the activation NMDA receptors and thereby has protective effect
on neurotoxicity and excitotoxicity following brain injury (Korani
et al., 2014). In silico study reported that GA form good binding
affinity with active sites of neurotoxic proteins, hydrogen bonding
with amino acid residues HIS A: 121, ARG B : 207 CYS A : 163, SER
A : 58, TYR B : 204 of caspase 3 ,GLN A: 156, ARG A: 40 , LYS A : 171
GLN : 175 of Interleukin 6, PHE B : 709, ALA B : 417, ARG B : 419,
ALA A : 417 , CYS A : 420 of Nitric oxide synthase, THR A: 501, PRO
A : 499, GLU A: 726, TYR A : 471, PRO A : 515, ARG B : 506 Gluta-
mate receptor. In silico studies showed good binding of gallic acid
with the caspase 3, Interleukin 6, Nitric oxide synthase and Gluta-
mate receptor and also exhibited good binding energy, it was
observed with marketed compounds such as Minocycline, Querce-
tin and Memantine, it is indicating it as a possible therapeutic
strategy for treatment of cerebral ischemic injury. GA inhibited
the expression of mitochondrial apoptotic signalling molecules
reported in various in vivo studies. Cerebral Ischemia stimulates
both the intrinsic pathway, which results from mitochondrial
crash-associated stimulation of caspase-9, and the extrinsic path-
way, which results from death receptor activation and subsequent
stimulation of caspase-8.(Sacco et al., 2009). Several studies
Fig. 8. Effect of Gallic acid on % of cerebral infarction Size in MO/RCA rats. revealed that protective effect of GA against oxidative stress, apop-
tosis, and inflammation (Abdel-Moneim et al., 2017; Maya et al.,
2018a, 2018b). GA has been reported to possess antioxidant, neu-
roprotective activities, in the treatment of depression (Nagpal
thickness of the pyramidal layer, with increased apoptotic neurons, et al., 2012) by acting on intracellular antioxidant enzymes such
decreased in the intact of neurons and irregular when compared to as superoxide dismutase (SOD) and catalase, by lowering
sham group. Moreover, Gallic acid treatment restore neuronal cells hydroperoxide through the glutathione peroxidase process
contact when compared with MO/RCA rats. Similarly, Cerebral (Nordberg and Arnér, 2001). In summary, our results demonstrate

Fig. 9. GA inhibiting MO/RCA increased Percent of Cerebral infract volume A) Normal rats B) Sham rats C) MO/RCA rats D) MO/RCA + GA 25 mg/kg rats E) MO/RCA + GA
50 mg/kg rats.

Table 5
Effect of Gallic acid on brain hippocampus biochemical parameters in MO/RCA injury rats.

Treatment Infarction size (%) GSH (nmol/mg) MDA (nmol/g) SOD (U/mg) Catalase(nmol/mg)
Normal rats 0 0.19 ± 0.01 6.43 ± 0.17 16.7 ± 0.18 0.0043 ± 0.00025
Sham rats 0 0.13 ± 0.01** 7.41 ± 0.15*** 13.5 ± 0.96** 0.0020 ± 0.00068**
MO/RCA rats 100% 0.10 ± 0.01*** 10.77 ± 0.99*** 9.8 ± 0.11*** 0.00022 ± 0.00005***
Gallic acid 25 mg/kg 22.03% 0.13 ± 0.01* 10.28 ± 0.18 ns 15.7 ± 0.42*** 0.00106 ± 0.00005***
Gallic acid 50 mg/kg 52.12% 0.18 ± 0.01*** 9.90 ± 0.55 ns 15.7 ± 0.42*** 0.0038 ± 0.00067***

Mean ± SEM were calculated for all data significant different between means where evaluated by One way Analysis Variance (ANOVA followed by t test comparison test
p < 0.05*, p < 0.01**, p < 0.001*** considered as statistical significant.

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P. Praveen Kumar, M. D., L. Siva Sankar Reddy et al. Saudi Journal of Biological Sciences 28 (2021) 5204–5213

Fig. 10. Effect of Gallic acid on brain biochemical parameters in MO/RCA rats.

Fig. 11. Effect of MO/RCA on brain hippocampus region of rats: The brain hippocampus region (x40) after induction of Cerebral stroke by MO/RCA. (A) Normal rats (B)
Sham rats C) MO/RCA D) MO/RCA + GA 25 mg/kg rats. E) MO/RCA + GA 50 mg/kg rats. In treated rats hippocampus region neuronal cells are less scattered, more neuronal cells
in contact (black arrow). Treated rats showing increased thickness of pyramidal cell layer in the hippocampus region, with decreased apoptotic neurons with dystrophic
changes in the form of shrunken and irregular (black arrows).

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P. Praveen Kumar, M. D., L. Siva Sankar Reddy et al. Saudi Journal of Biological Sciences 28 (2021) 5204–5213

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